The use of optically detectable labeling groups, and particularly those groups having high quantum yields, e.g., fluorescent or chemiluminescent groups, is ubiquitous throughout the fields of analytical chemistry, biochemistry and biology. In particular, by providing a highly visible signal associated with a given reaction, one can better monitor that reaction as well as any potential effectors of that reaction. Such analyses are the basic tools of life science research in genomics, diagnostics, pharmaceutical research, and related fields.
To date, such analyses have generally been performed under conditions where the amounts of reactants are so far in excess that any adverse effects on the optical event are unnoticed. For example, such analyses based upon fluorescent labeling groups generally require the use of an excitation radiation source, e.g., a light source, directed at the reaction mixture, to excite the fluorescent labeling group, which is then separately detectable. However, prolonged exposure of chemical and biochemical reactants to such light sources, alone, or when in the presence of other components, e.g., the fluorescent groups, can lead, potentially, to damage to such reactants, e.g., proteins, enzymes, substrates, or the like.
Fluorescence is the result of a three-stage-process that occurs in the fluorophores or fluorescent dyes. The three-stage process includes: 1) excitation in which a photon with quantized energy from an external light source with certain wavelength is supplied and absorbed by the fluorophore, creating an excited electronic singlet state (S1′); 2) excited-state lifetime, during which the excited fluorophore undergoes several different changes to relax its energy to the lowest singlet state (S1); and 3) fluorescence emission in which a photon of energy (S1-S0) is emitted returning the fluorophore to its ground state.
One of the many pathways that dissipate the energy of the excited electronic singlet state is the intersystem crossing (ISC), involving a change in spin multiplicity, transiting the electron from S1 to the excited triplet state (T1). In many fluorescent dye molecules the formation of the much longer life-time triplet-state species greatly reduced the brightness of the fluorescence emission. In addition, it exhibits a high degree of chemical reactivity in this state, which often results in photobleaching and the production of damaging free radicals.
As noted previously, however, conventional formats for such reactions generally prevent any such effects from being problematic, or even being noticed.
A variety of analytical techniques are being explored, however, that deviate from previous formats, such that detrimental effects of such photo-induced damage have a more dramatic impact on the operation of the given analysis. In particular, real-time analyses of reactions that include fluorescent reagents can expose multiple different components to optical energy. Additionally, reactions based upon increasingly smaller amounts of reagents, e.g., in microfluidic or nanofluidic reaction vessels or channels, or in “single molecule” analyses, are more severely impacted by such damage. As such, the present invention is directed at methods and compositions that prevent or mitigate to some extent, the adverse effects of such photo-induced damage, and also to processes that benefit from such methods and/or compositions, among other useful processes and compositions.